Co-evolution of genetically encoded light-up aptamers and fluorogenic probes for live-cell super-resolution RNA imaging

完整细胞中的RNA成像以高时间和空间分辨率揭示转录、翻译、基因表达和调控的过程。不幸的是，由于缺乏固有的荧光RNA，体内RNA的成像和检测一直受到阻碍。现有的点亮适体仍然缺乏对单个RNA分子进行高空间和时间分辨率的体内成像的灵敏度。我们的目标是开发具有单分子灵敏度的超分辨率RNA成像技术，使其能够在体内研究RNA生物化学。在第一个资助期取得的进展的基础上，我们的目标是：i)利用SELEX和FACS技术的组合来进化SIRA和SRB-4适配子，以改善荧光开启因子和解离常数。ii)合成基于Janelia染料的新一代罗丹明类荧光团，具有高量子产率和光稳定性。Iii)使用SIRA和SRB-4发光适配子结合使用超分辨率显微镜(STED和STORM)的新探针来成像活细胞中不同的RNA分子。iv)利用FRET概念来提高荧光显微镜中RNA检测的灵敏度。(SRB-4和Sira不仅相互正交，而且是一对很好的FRET)。V)探索不同的概念(即旋转猝灭和变色荧光团)以产生新类别的发光适配子。Vi)开发除非与适配子靶标结合否则不会发出荧光的超暗探针。vii)利用单分子RNA成像的“亲和力”概念，其中将使用新的RNA支架来预先组织适配子二聚体以用于探针结合。

Imaging RNA in intact cells reveals the processes of transcription, translation, gene expression and regulation with high temporal and spatial resolution. Unfortunately, the imaging and detection of RNA in vivo has been hampered by the lack of intrinsically fluorescent RNAs. The currently available light-up aptamers still lack the sensitivity to image single RNA molecules with high spatial and temporal resolution in vivo. Our goal is to develop super-resolution RNA imaging techniques with single-molecule sensitivity that allow studying RNA biochemistry in vivo. Based on the progress made in the first funding period, we aim to:i) evolve SiRA and SRB-4 aptamers using a combination of SELEX and FACS techniques to improve the fluorescence turn-on factors as well as dissociation constants.ii) synthesize next-generation rhodamine-based fluorophores with high quantum yields and phostability based on Janelia dyes for SiRA, SRB-4, and their variants. iii) image distinct RNA molecules in live cells using SiRA and SRB-4 light-up aptamers combined with new probes using super-resolution microscopy (STED and STORM).iv) utilize the FRET concept to improve the sensitivity for RNA detection in fluorescence microscopy. (SRB-4 and SiRA are not only orthogonal to each other, but also a good FRET pair). v) explore different concepts (namely rotational quenching and color-shifting fluorophores) to generate new classes of light-up aptamers. vi) to develop super-dark probes which do not fluorescence unless they bind to the aptamer target.vii) utilize the “avidity” concept for single-molecule RNA imaging, where new RNA scaffolds will be employed to pre-organize aptamer dimers for probe binding.